Peptide having ability to activate cancer-related gene

ABSTRACT

To provide a cancer diagnostic reagent for determining malignancy of a cancer patient or a cancer cell and a tendency of canceration of a healthy subject, the reagent including a peptide having an ability to activate a cancer-related gene and extracted from cell membrane surfaces of human squamous-cell carcinoma cells or including a synthetic polynucleotide encoding the peptide or a partial amino acid sequence of the peptide.

This application is a division of U.S. patent application Ser. No.11/486,123 filed on Jul. 14, 2006, now U.S. Pat. No. 7,579,434 issuedAug. 25, 2009, which is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to peptides activating a cancer-relatedgene and derived from cell membrane surfaces of human cancer cells,immunosuppressive agents including the peptides as an effectivecomponent, and anticancer agents including antibodies against thepeptides as an effective component. The present invention furtherrelates to diagnostic reagents including polynucleotides encoding thepeptides for diagnosing malignancy of cancer or tendency of canceration.Furthermore, the present invention relates to methods for manufacturingthe peptides.

2. Description of the Related Art

Cancer is a disease caused by cells which started unregulated growth.The cells continue to proliferate and infiltrate into neighboring normalcells to destroy the normal functions. Some of the cells spread bymetastasis from their original site to one or more sites elsewhere inthe body, leading to a loss of normal cell functions and to depressionof functions of organs. Thus, cancer is a disease that leads patients todeath. Normal cells proliferate but will stop growing when they come incontact with solid substances (contact inhibition). On the other hand,the contact inhibition property is lost in cancerous cells.Consequently, the cancerous cells continue to grow in their host as longas the host is alive. Among cells extracted from the body, cells havingability for continuing unlimited growth can be established as a cellline. Cancer, in a broad sense, is a state of uncontrolled cell growthdue to mutation of a gene, in particular, due to mutation of a genepositively or negatively regulating cell growth. Heretofore, manystudies have been conducted on genes contributing to carcinogenesis or agrowth mechanism and peptides or proteins relating to them (JapaneseUnexamined Patent Application Publication Nos. 2003-517306 and2000-217585).

Among peptides or proteins derived from cell membrane surfaces of humancancer cells, generally, HLA-binding peptides are particularly thoughtto be cancer antigens and are thought to act on immunocompetent cells asantigens, most of all, as immunogens. Additionally, it is widely knownthat some proteins and glycoproteins derived from cancer cells andimmunocompetent cells such as macrophages isolated from a cancer-bearingliving body have an immunosuppressing activity preventing thedestruction of cancer cells, for example, immunosuppressive acidicprotein (IAP). Such proteins and glycoproteins are clinically used formeasuring the degree of immunosuppression.

Heretofore, it has been thought that cancer cell growth progresses by amechanism due only to self division of cancerous cells. However, therate of cancer cell growth is not constant and cancer cells rapidlyproliferate at some point. Such phenomena cannot be fully explained bythe conventional view only.

SUMMARY OF THE INVENTION

On the basis of the above-mentioned phenomenon that cancer rapidlyprogresses at some point, cancer cells have a possibility of secreting afactor outside the cancer cells. Such a factor further accelerates thecancer cell growth by acting on normal cells neighboring the cancercells so as to activate a cancer-related gene in the normal cells. Ifthis factor is found, not only a mechanism of progression of cancer isclarified but also a useful drug can be provided.

Namely, it is an object of the present invention to provide a factorhaving a function of accelerating cancer cell growth by acting on normalcells neighboring the cancer cells so that a cancer-related gene in thenormal cells is activated and to provide a corresponding gene of thefactor. It is another object of the present invention to develop avaluable use of the factor and its corresponding gene by verifying thefactor for the cancer-related-gene-activating function in normal cells.

The present inventor has performed intensive studies for overcoming theabove-mentioned problems, and, as a result, found a peptide and a geneencoding the peptide in an extract of a human squamous-cell carcinomacell line UTC-8 which has a high activity of metastasis. Furthermore,the inventor has proved that the peptide has a function of activating acancer-related gene in normal cells neighboring cancer cells by actingon the normal cells and accelerating cancer growth, and has proved thatthe peptide can be used as an immunosuppressive agent. In addition, theinventor has found that a polynucleotide capable of hybridizing with thegene encoding the peptide can be used as a reagent for diagnosingmalignancy of cancer or measuring tendency of canceration (easiness ofconversion to cancer: a degree of risk of canceration), which was notincluded in clinical examination items before, and that an antibodyagainst the peptide has an anticancer activity. Thus, the presentinvention has been accomplished.

The present invention relates to aspects (1) to (10) described below:

(1) A peptide having an ability to activate a cancer-related gene,wherein the peptide is derived from cell membrane surfaces of humansquamous-cell carcinoma cells and includes an amino acid sequencerepresented by SEQ ID No: 1 or an amino acid sequence having deletion,substitution, or addition of one or several amino acids in the aminoacid sequence represented by SEQ ID NO: 1;

(2) A peptide having an ability to activate a cancer-related gene,wherein the peptide is derived from cell line UTC-8 (FERM BP-08611)established from human squamous-cell carcinoma; and the peptide shows adetectable peak at a detection wavelength of 214 nm in gel filtration ofan extract from the cell line;

(3) An immunosuppressive agent comprising the peptide according to theaspect (1) or (2) as an effective component;

(4) An antibody against the peptide according to the aspect (1) or (2);

(5) A diagnostic reagent including the antibody according to the aspect(4) for determining tendency of canceration or malignancy of cancer;

(6) An anticancer agent comprising the antibody according to the aspect(4) as an effective component;

(7) A polynucleotide including a nucleotide sequence encoding at leastthree contiguous amino acids of the amino acid sequence of the peptideaccording to the aspect (1);

(8) A diagnostic kit for determining tendency of canceration ormalignancy of cancer, wherein the kit includes a peptide according tothe aspect (1) or (2) and a polynucleotide according to the aspect (7);

(9) The diagnostic kit according to the aspect (8) further including atleast one marker gene selected from the group consisting of:

Ras oncogene family;

v-crk avian sarcoma virus CT10 oncogene homolog-like lactatedehydrogenase B;

Placental growth factor;

Interleukin 8;

MAS1, activator of S phase kinase;

v-raf;

v-fms;

v-rel;

v-src;

GRO1;

Hepatoma-derived growth factor;

Vascular endothelial growth factor;

Bone morphogenic protein 3;

Squamous-cell carcinoma antigen recognized by T cell;

Interleukin-1 beta;

Conserved gene amplified in osteosarcoma; and

Lymphoid blast crisis oncogene; and

(10) A method for preparing a peptide according to the aspect (1) or(2), the method including the steps of immersing cells obtained byculturing human squamous-cell carcinoma cells in a culture medium into acitrate-phosphate buffer having a pH of 3.3 to 3.4 for extraction; andfractionating the extract by gel filtration using a citrate-phosphatebuffer having a pH of 6.8 to 7.0 for yielding a peptide having anability to activate a cancer-related gene in human normal cells.

The peptide according to the present invention is derived from cellmembrane surfaces of human squamous-cell carcinoma cells. In particular,the peptide is extremely remarkable in the fact that it has ability toactivate a cancer-related gene in human normal cells. Since thesensitivity of cells and tissues against this peptide of the presentinvention can be an indicator to predict canceration tendency of normalcells or progress of cancer, the peptide can be a useful diagnosticreagent for determining tendency of canceration or malignancy of cancer.Additionally, it is strongly suggested that the unregulated growth ofcancer cells is caused by, not only cancer cells themselves, but alsothe interaction accompanying the activation of various cancer-relatedgenes in normal cells. Thus, an entirely novel mechanism of cancergrowth is presented.

Therefore, the present invention can contribute greatly to the researchand development of diagnosis and therapy of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are elution patterns of gel filtration of peptidesextracted in Example 1 according to the present invention. FIG. 1A showselution patterns of the peptides by using a citrate-phosphate bufferhaving a pH of 7.2 as an elution buffer and detection wavelengths of 280nm and 210 nm. FIG. 1B shows elution patterns of the fraction (elutionvolume: 21 ml) of the rightmost peak of the elution pattern using thedetection wavelength of 280 nm in FIG. 1A by using a citrate-phosphatebuffer having a pH of 6.8 as an elution buffer and detection wavelengthsof 280 nm and 214 nm.

FIGS. 2A and 2B are electrophoresis photographs showing gene patternchanges in cells treated with a peptide of the present invention whichwere investigated by conducting a PCR reaction using cDNA prepared byreverse transcription of total mRNA derived from the cells treated withthe peptide as a template and using DNA encoding the full length of thepeptide of the present invention as a primer.

FIGS. 3A and 3B are electrophoresis photographs showing gene patternchanges in cells treated with a peptide of the present invention. Thegene was prepared by a PCR reaction using cDNA prepared by reversetranscription of total mRNA derived from the cells treated with thepeptide as a template and using DNA encoding a sequence consisting ofthe first to seventh amino acids of the peptide of the present inventionas a primer.

FIG. 4 is a photograph showing the result when the monoclonal antibodyagainst the peptide of the present invention was subjected to acytotoxic test using cancer cells.

FIG. 5 is a diagram showing an immunosuppressive activity of the peptideof the present invention in a transplantation test.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The peptide according to the present invention has a function ofactivating a human cancer-related gene and includes the following aminoacid sequence:

Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu-Ser-Lys (SEQ ID NO: 1)

Additionally, a peptide including an amino acid sequence havingdeletion, substitution, or addition of one or several amino acids in theamino acid sequence represented by SEQ ID NO: 1 is included in thepresent invention as long as the peptide has the function of activatinga cancer-related gene.

The above-mentioned cancer-related gene refers to not only oncogenes butalso immune system genes. The activation of a cancer-related gene meansacceleration of canceration or cancer growth through activation of anoncogene or through inactivation of a tumor suppressor gene and/oractivation or inactivation of an immune system gene, e.g., a cellularimmunity system and/or humoral immunity system.

The immunosuppressive activity of the peptide according to the presentinvention is notable. For example, it is recognized that the peptidesuppresses the rejection in tissue transplantation. Therefore, thepeptide of the present invention can be used as an immunosuppressiveagent.

The peptide represented by SEQ ID NO: 1 is derived from cell membranesurfaces of human squamous-cell carcinoma cells and can be extractedfrom human squamous-cell carcinoma cell line UTC-8 (Deposition No.: FERMBP-08611, which is deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, Central 6, 1-1-1 Higashi, Tsukuba-shi, Ibaraki-ken,305-8566, Japan, on Feb. 4, 2004) by using a citrate-phosphate bufferhaving a pH of 3.3 to 3.4.

More specifically, after the extraction by the citrate-phosphate bufferhaving a pH of 3.3 to 3.4, the extract is further applied to gelfiltration using a citrate-phosphate buffer having a pH of 6.8 to 7.2 asan elution buffer at a column flow rate of 0.1 to 0.28 ml/min, and thena fraction having a peak in a detection wavelength of 214 nm is isolatedto yield the peptide represented by SEQ ID NO: 1.

The peptide represented by SEQ ID NO: 1 of the present invention ischaracterized by the fact that the peak can be separated and detectedonly when a detection wavelength of 214 nm is used. The peak of thepeptide of the present invention cannot be detected when a detectionwavelength of 254 to 257 nm or of 280 nm, which are generally used inpeptide detection, is used.

In addition to the above-mentioned extraction method, the peptideaccording to the present invention can be prepared by conventionalchemical synthesis of peptides on the basis of the amino acid sequence.Namely, an amino acid derivative having a carboxyl group and aside-chain functional group being protected by a protecting group and anamino acid derivative having an amino group and a side-chain functionalgroup being protected by a protecting group are condensed in thepresence of carbodiimide or the like. Then, the protecting group for theamino group is removed to bind a next amino acid derivative protected bya protecting group. Such a reaction can be performed by a liquid-phasemethod or a solid-phase method. In general, the solid-phase method isused, except when a relatively large amount of peptide is synthesized.Particularly, in a method developed for identifying an antigenicdeterminant defined by its amino acid sequence, a spacer arm having anamino group on its tip is used. The spacer arm is prepared by chemicallytreating a cellulose film or the tip of a plastic pin. In the lattermethod, a peptide chain is extended by sequential reactions of solutionsin a 96-well plate; this method is called multi-pin peptide synthesis.

Furthermore, in another method, a peptide of the present invention maybe prepared by chemically synthesizing a DNA encoding the peptide byusing a DNA synthesizer; preparing a recombinant vector by connectingthe DNA to an appropriate expression vector; introducing the vector intoa host such as Escherichia coli; culturing the host; and collecting thepeptide of the present invention from the culture.

The peptide of the present invention is useful by itself and can be usedas a diagnostic reagent for determining tendency of canceration(easiness of conversion to cancer: a degree of risk of canceration) ofnormal cells or tissues, for determining whether or not a subject issuffering from cancer, and for determining malignancy of cancer.

The above-mentioned various determinations are performed by bringing thepeptide of the present invention in contact with a human cell or tissuespecimen obtained by surgical resection or biopsy, and utilizing thiscontact for detecting a cancer-related gene or for determining a changein the expression degree or the expression pattern of the gene. Thesensitivity of cells or tissues against the peptide of the presentinvention reflects, for example, a tendency of canceration (easiness ofconversion to cancer: a degree of risk of canceration) of the cells ortissues or a degree of malignancy of cancer.

In the determination of the degree of malignancy of cancer cells ortissues, the amount of expression of the gene encoding the peptide ofthe present invention may be directly determined without the contact ofthe peptide of the present invention with the cells or tissues. However,it is desirable that the cancer cells or tissues are brought intocontact with the peptide of the present invention, in order to achievehigher sensitivity in measurement.

Additionally, as is clear in diagnosis methods 1 and 2 described below,a polynucleotide (DNA and RNA) encoding at least three contiguous aminoacids of the amino acid sequence of the peptide of the present inventioncan be used as a diagnostic reagent for determining tendency ofcanceration (easiness of conversion to cancer: a degree of risk ofcanceration) of normal cells or tissues, for determining whether or nota subject is suffering from cancer, and for determining malignancy ofcancer. A diagnostic kit of the present invention preferably utilizessuch a polynucleotide in combination with a peptide of the presentinvention.

Diagnosis methods using the peptide of the present invention will now bedescribed.

[Diagnosis Method 1]

First, a specimen such as cells and tissues obtained from a subject tobe tested is brought into contact with the peptide of the presentinvention, and they are cultured for a predetermined period of time.Examples of the specimen include cells and tissues obtained from a highrisk group for cancer, such as subjects living in area polluted with achemical or radioactive material and smokers; cells and tissues whichare suspected to be a precancerous stage, such as polyp; cells andtissues neighboring cancer cells; cells and tissues at a region under adanger of metastasis of cancer; and peripheral blood.

When the specimen has a tendency of canceration, even if the cells andtissues are normal now, a change in an expression pattern of the gene isobserved: the gene encoding the peptide of the present invention isdetected, or the amount of expression of the gene is increased. When themalignancy of cancer is high, the amount of expression of the gene isfurther increased. Therefore, the tendency of canceration or malignancyof cancer can be determined by detecting the gene or determining thedegree of its expression.

In this diagnosis method, the cells or tissues of interest are broughtinto contact with the peptide, and then total mRNA is extracted from thecells or tissues. After synthesis of cDNA by reverse transcription usingthe mRNA as a template, PCR is further conducted by using the resultingcDNA as a template. In the latter PCR, all polynucleotides encodingsequences consisting of at least three contiguous amino acid residues ofthe amino acid sequence represented by SEQ ID NO: 1 are collectivelyused as primers. The polynucleotide may encode the full-lengthpolypeptide.

This diagnosis method will now be specifically described.

First, total mRNA is extracted by a common method from a specimen whichis prepared by treating normal tissues or cells with the peptide. Then,cDNA is synthesized by reverse transcription using the total mRNA astemplates to generate a cDNA pool which will be used as templates.

DNA encoding a sequence consisting of, for example, the first to fourthamino acids in the amino acid sequence represented by SEQ ID NO: 1 hasthe following nucleotide sequence:

CAR CCN CAR TTY (SEQ ID NO:2)

(wherein N is A, T, C, or G, R is G or A, and Y is C or T). Each of thenucleotide sequences, i.e., 32 types of DNA, is synthesized, and a poolincluding each DNA in an equal amount is used as forward primers.

Since the cDNA used as the template has a poly-T sequence correspondingto poly-A tailing of mRNA, an oligo-dT primer (poly-A primer) is used asa reverse primer.

The PCR using the cDNA pool as the templates is conducted by using theabove-mentioned forward primers and the reverse primer, followed byelectrophoresis and fluorescence staining.

When a change in an expression pattern of genes including the geneencoding the peptide of the present invention is induced by treating aspecimen with the peptide of the present invention, it is indicated thatthe specimen is sensitive to the treatment with the peptide of thepresent invention; thus, it is determined that the specimen has a hightendency of canceration even if the cells or tissues as the specimen arecurrently normal. When a specimen is already cancerous, a change in thegene expression pattern increases with a degree of the malignancy.Therefore, the degree of the malignancy can be determined by measuringthe change.

The amount of the peptide expression can be determined by measuring thefluorescence intensity of the band in the electrophoresis. In order toprecisely determine the amount of the peptide expression, for example,quantitative PCR may be performed.

The quantitative PCR can be performed by, for example, real-time PCRusing the cDNA pool as templates, the above-mentioned primers, and afluorescent dye or a fluorescence-labeled probe which binds withdouble-strand DNA. Then, a relationship between the number of the PCRcycles and the fluorescence intensity is determined. By comparing theresults with those obtained by using a standard, the amount of the cDNA,i.e., the expression amount of the peptide of the present invention, canbe quantitatively determined.

A change in the gene expression pattern or an increase in the peptideexpression amount can be determined by comparing the expression level ofa control. As the control, the results obtained by conducting the sameprocedure as above using the same cells except that the peptide of thepresent invention is not used and/or the results obtained by treatingnormal cells or tissues that are completely free from cancer risk withthe peptide and conducting the same procedure as above, are used.

The normal cells and tissues that are free from cancer risk are derivedfrom young subjects. As such cells, for example, a kidney mesangium cell(ACBRI-1376; Applied Cell Biology Research Institute), a skin fibroblastcell (Catlog 2F0-C25; Cell Systems), and a pancreatic epithelial cell(CBRI515; Applied Cell Biology Research Institute) are commerciallyavailable.

From the views described above, it is obvious that a combination of thepeptide of the present invention and the group of the above-mentionedprimers, i.e., the group of polynucleotides encoding sequencesconsisting of at least three contiguous amino acid residues of the aminoacid sequence represented by SEQ ID NO: 1, is useful as a cancerdiagnostic reagent kit for determining a tendency of canceration ormalignancy of cancer.

[Diagnosis Method 2]

In another diagnosis method using the peptide of the present invention,cancer-related gene is used as a marker gene.

In this diagnosis method, cells or tissues of interest are treated withthe peptide of the present invention as in the above-mentioned diagnosismethod 1; thus the same specimen as in the diagnosis method 1 can beused. However, in this diagnosis method, the reverse transcription isconducted to generate a cDNA (first-strand cDNA) pool, which iscomplementary to the mRNA, by using total mRNA extracted from the cellsor tissues treated with the peptide as a template and using oligo-dTprimer having T7 RNA polymerase promoter region as a reverse primer.Then, after the second-strand cDNA synthesis, in vitro transcription(IVT) was conducted using T7 RNA polymerase and biotinylated rNTPs at37° C. for 14 hr to yield biotin-labeled cRNA. The amount of the cRNA ismeasured with a spectrophotometer to confirm that at least 10 μL of cRNAcan be yielded.

The diagnosis method further includes processes for bringing this cRNAlabeled with fluorescence dye or the like into contact with cDNA of acancer-related gene which is immobilized on a plate for hybridizationand for measuring the fluorescence intensity.

When the cRNA is hybridized to some cancer-related gene, it is suggestedthat the cancer-related gene is being expressed in the cells or tissues.When the fluorescence intensity of some cancer-related gene is increasedor decreased, it is suggested that the expression amount of thecancer-related gene is increased or decreased by the treatment with thepeptide of the present invention. Namely, for example, when theexpression of an oncogene is increased or the expression of an immunesystem gene which suppresses canceration is decreased, it is suggestedthat the tendency of canceration is high. Thus, on the basis of theexpression condition of a cancer-related gene, a tendency of cancerationor malignancy of cancer can be determined.

An increase or decrease in the expression amount of a cancer-relatedgene is determined by comparing the expression level in a control. Thecells and tissues used as the control are the same as those used in thediagnosis method 1.

As described above, immune system genes relating to cancer, in additionto oncogenes, are included in the cancer-related genes in thisspecification. Examples of such genes are as follows:

Ras oncogene family;

v-crk avian sarcoma virus CT10 oncogene homolog-like lactatedehydrogenase B;

Placental growth factor;

Interleukin 8;

MAS1, activator of S phase kinase;

v-raf;

v-fms;

v-rel;

v-src;

GRO1;

Hepatoma-derived growth factor;

Vascular endothelial growth factor;

Bone morphogenic protein 3;

Squamous-cell carcinoma antigen recognized by T cell;

Interleukin-1 beta;

Conserved gene amplified in osteosarcoma; and

Lymphoid blast crisis oncogene.

At least one gene of these cancer-related genes is used in thisinvention. For higher precision, yet more cancer-related genes may beused. In such a case, it is preferable to use a combination of genes inthe following groups as a genes set for diagnosis.

(a) Oncogene expression-increasing group: a gene encoding the peptide ofthe present invention and Ras oncogene and c-fos oncogene, etc.;

(b) Immune gene expression-increasing group: Interleukin-1 beta, etc.;and

(c) Immune gene expression-decreasing group: MHC class II, DM, andalpha, beta protein gene and killer cell lectin-like receptor subfamilyB, M member 1 gene.

Tables 1 and 2 show examples of cancer-related genes of which expressionis increased and immune system genes of which expression is increased ordecreased by the treatment with the peptide (HPLC-purified peptide)derived from cell membrane surfaces of human squamous-cell carcinomacells UTC-8 (Deposition No.: FERM BP-08611, which is deposited withInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology).

TABLE 1 Cancer-related gene enhanced by the HPLC- purified peptide GeneName Fold Change Common Description Product Phenotype Function KeywordsK03218 3.912448639 SRC; v-src proto-oncogene Colon c-myc ASV; sarcomatyrosine-protein cancer, proto- SRC1; (Schmidt- kinase SRC advancedoncogene; c-SRC; Ruppin A-2) proto- p60-Src viral oncogene; oncogene srcgene; homolog src (avian) oncogene NM_001201 3.73617959 BMP3 bone bonemorphogenetic morphogenetic protein 3 (osteogenic) protein 3 precursor(osteogenic) D16431 3.595973492 HDGF hepatoma- hepatoma-derivedhepatoma- derived growth factor (high- derived growth mobility groupprotein GF; factor 1-like) hepatoma- (high- derived mobility growthgroup factor protein 1- like) X54936 3.576227427 PGF placental placentalgrowth placenta growth factor, vascular growth factor, endothelialgrowth factor vascular factor-related protein endothelial growth factor-related protein M95712 3.033826351 BRAF v-raf murine v-raf murinesarcoma Adenocarcinoma b-raf sarcoma viral oncogene homolog of oncogene;viral B1 lung, serine/threonine oncogene somatic; protein homolog B1Colorectal kinase cancer, somatic; Melanoma, melignant, somatic;Nonsmall cell lung cancer, somatic NM_005850 3.023255825 SF3B4 splicingsplicing factor 3b, factor 3b, subunit 4 subunit 4, 49 kDa NM_0059372.995664358 MLLT6 myeloid/lymphoid myeloid/lymphoid or or mixed-lineageleukemia mixed- (trithorax homolog, lineage Drosophila); leukemiatranslocated to, 6 (trithorax homolog, Drosophila); translocated to, 6NM_001419 2.950166225 ELAVL1 ELAV ELAV-like 1 (embryonic lethal,abnormal vision, Drosophila)- like 1 (Hu antigen R) NM_002613 2.86612194PDPK1 3- 3-phosphoinositide protein phosphoinositide dependent proteinkinase dependent kinase-1 protein kinase-1 NM_014308 2.849243879 P101-phosphoinositide- phosphoinositide-3- PI3K 3- kinase, regulatory kinase,subunit, polypeptide regulatory p101 subunit, polypeptide p101 D261202.833239079 splicing factor 1 X61498 2.832505941 NFKB2 nuclear nuclearfactor of NF-kb factor of kappa light subunit kappa light polypeptidegene polypeptide enhancer in B-cells 2 gene (p49/p100) enhancer inB-cells 2 (p49/p100) X03663 2.780905962 CSF1R colony colony stimulatingMyeloid c-fms stimulating factor 1 receptor malignancy, oncogene; factor1 precursor predisposition fms receptor, to oncogene; formerlyglycoprotein; McDonough membrane feline protein; sarcoma proto- viral(v- oncogene; fms) signal oncogene peptide homolog NM_004204 2.756244421PIGQ phosphatidylinositol phosphatidylinositol glycan, glycan, class Qclass Q isoform 2; phosphatidylinositol glycan, class Q isoform 1NM_003017 2.741738006 SFRS3 splicing splicing factor, factor,arginine/serine-rich 3 arginine/serine- rich 3 NM_000753 2.7408638 PDE3Bphosphodiesterase phosphodiesterase 3B, 3B, cGMP-inhibited cGMP-inhibited NM_002712 2.696629047 PPP1R7 protein protein phosphatase 1,regulatory phosphatase regulatory subunit 7 polypeptide 1, of regulatoryprotein subunit 7 phosphatase-1 NM_004906 2.543901205 WTAP Wilms tumorWilms' tumour 1- 1 associated associating protein protein isoform 1;Wilms' tumour 1-associating protein isoform 2 NM_005207 2.497231245 CRKLv-crk v-crk sarcoma virus sarcoma CT10 oncogene homolog virus CT10(avian)-like oncogene homolog (avian)-like NM_007279 2.493814707 U2AF2U2 small U2 small nuclear nuclear ribonucleoprotein ribonucleoproteinauxiliary factor auxiliary (65 kD) factor (65 kD) NM_015714 2.480620146G0S2 putative putative lymphocyte lymphocyte G0/G1 switch gene G0/G1switch gene NM_001511 2.476593256 CXCL1 chemokine chemokine (C—X1—C(C—X—C motif) ligand 1 motif) ligand 1 (melanoma growth stimulatingactivity, alpha) NM_016263 2.465854406 FZR1 Fzr1 protein Fzr1 proteinNM_012103 2.459948301 AUP1 ancient ancient ubiquitous ubiquitous protein1 isoform 1; protein 1 ancient ubiquitous protein 1 isoform 2; ancientubiquitous protein 1 isoform 3 M32977 2.456767944 VEGF vascular vascularendothelial Diabetic angiogenic endothelial growth factor retinopathy,mitogen; growth NIDDM- vascular factor related, endothelialsusceptibility growth to factor NM_007040 2.432915688 E1B-AP5 E1B-55kDa- E1B-55 kDa-associated associated protein 5 isoform a; protein 5E1B-55 kDa-associated protein 5 isoform d; E1B-55 kDa-associated protein5 isoform b; E1B-55 kDa-associated protein 5 isoform c NM_0014022.404632807 EEF1A1 eukaryotic eukaryotic translation translationelongation factor 1 elongation alpha 1 factor 1 alpha 1 NM_0023572.396934271 MAD MAX MAX dimerization dimerization protein 1 protein 1NM_005524 2.39485383 HES1 hairy and hairy and enhancer of enhancer ofsplit 1 split 1, (Drosophila) NM_005730 2.387011766 CTDSP2 conservednuclear LIM interactor- gene interacting factor 2 amplified inosteosarcoma AF040963 2.356589317 MXD4 MAX MAD4 dimerization protein 4M83221 2.351067066 RELB v-rel reticuloendotheliosis NF-kappa-B I-Rel;reticuloendotheliosis viral oncogene homolog B transcpription NF-kappa-Bviral factor transcription oncogene p50- factor homolog B, subunitinhibitor nuclear inhibitor factor of kappa light polypeptide geneenhancer in B-cells 3 (avian) NM_006842 2.24469161 splicing factor 3b,subunit 2, 145 kDa NM_001570 2.239902258 IRAK2 interleukin-1interleukin-1 receptor- receptor- associated kinase 2 associated kinase2 NM_006716 2.230443954 ASK activator activator of S phase of S phasekinase kinase NM_004723 2.209302187 ARHGEF2 rho/rac rho/rac guanineguanine nucleotide exchange nucleotide factor 2 exchange factor (GEF) 2M60119 2.204995632 HIVEP2; human human immunodeficiency MBP-2;immunodeficiency virus type I enhancer HIV-EP2 virus binding protein 2type I enhancer binding protein 2 NM_001665 2.111301422 ARHG ras homologras homolog gene gene family, member G (rho family, G) member G (rho G)M62829 2.104651213 EGR1 early early growth response 1 transcriptiongrowth factor response 1 NM_004218 2.102017164 RAB11B RAB11B, RAB11B,member RAS member RAS oncogene family oncogene family NM_0063182.098191261 putative glialblastoma cell differentiation- related U338192.0930233 MAZ MYC- MYC-associated zinc associated finger protein zincfinger protein (purine- binding transcription factor) M13150 2.069767475MAS1 MAS1 MAS1 oncogene mas oncogene oncogene; mas protein; membraneprotein; proto- oncogene NM_003685 2.039983511 KHSRP KH-type KH-typesplicing splicing regulatory protein regulatory (FUSE binding proteinprotein 2) (FUSE binding protein 2) NM_006694 2.018802404 JTB; jumpingjumping translocation PAR; translocation breakpoint hJT; breakpointHJTB; B PAR NM_006191 2.002365112 PA2G4 proliferation- proliferation-associated associated 2G4, 38 kDa 2G4, 38 kDa X78710 2.001409531 MTF1metal- metal-regulatory metal- regulatory transcription factor 1regulatory transcription transcription factor 1 factor; MTF-1 gene;transcription factor

TABLE 2 Immune system gene regulated by the HPLC-purified peptide GeneName TDP Description Product Phenotype function Keywords Immune systemgene enhanced by the peptide 1 K02770 11.69 interleukin 1, betainterleukin 1, beta interleukin- interleukin; proprotein 1 receptorinterleukin 1 binding; signal transducer activity 2 J04130 7.341chemokine (C-C motif) chemokine (C-C receptor act2 ligand 4 motif)ligand 4 signaling gene; precursor protein immune tyrosine activationkinase gene activity; chemokine activity 3 NM_000211 3.326 integrin,beta 2 integrin beta chain, Leukocyte cell (antigen CD18 (p95), beta 2precursor adhesion adhesion lymphocyte function- deficiency receptorassociated antigen 1; activity macrophage antigen 1 (mac-1) betasubunit) 4 X03663 2.781 colony stimulating colony stimulating Myeloidmacrophage c-fms factor 1 receptor, factor 1 receptor malignancy, colonyoncogene; formerly McDonough precursor predisposition stimulating fmsfeline sarcoma viral to factor oncogene; (v-fms) oncogene receptorglycoprotein; homolog activity; membrane ATP binding; protein;transferase proto- activity oncogene; signal peptide 5 NM_006186 2.06nuclear receptor nuclear receptor Parkinson steroid subfamily 4, groupA, subfamily 4, group disease hormone member 2 A, member 2 isoformreceptor a; nuclear receptor activity; subfamily 4, group transcriptionA, member 2 isoform factor b; nuclear receptor activity subfamily 4,group A, member 2 isoform c; nuclear receptor subfamily 4, group A,member 2 isoform d 6 NM_002000 1.871 Fc fragment of IgA, Fc alphareceptor receptor receptor for isoform a precursor; activity; Fc alphareceptor receptor isoform b precursor; signaling Fc alpha receptorprotein isoform c precursor; activity Fc alpha receptor isoform d; Fcalpha receptor isoform e; Fc alpha receptor isoform f; Fc alpha receptorisoform g; Fc alpha receptor isof 7 NM_003199 1.621 transcription factor4 transcription factor RNA 4 isoform b polymerase II transcriptionfactor activity; DNA binding 8 NM_001733 1.543 complement componentcomplement component C1r/C1s complement 1, r subcomponent 1, rsubcomponent deficiency, component combined C1r activity; trypsinactivity; calcium ion binding; chymotrypsin activity; hydrolase activity9 NM_002983 1.542 chemokine (C-C motif) chemokine (C-C chemokine ligand3 motif) ligand 3 activity; antiviral response protein activity; signaltransducer activity 10 NM_002969 1.53 mitogen-activatedmitogen-activated MAP kinase protein kinase 12 protein kinase 12activity; ATP binding; protein serine/threonine kinase activity;transferase activity; SAP kinase 3 activity 11 NM_000173 1.529glycoprotein Ib platelet Bernard- thrombin (platelet), alphaglycoprotein Ib Soulier receptor polypeptide alpha polypeptide syndromeactivity; precursor cell adhesion molecule activity 12 D10202 1.506platelet-activating platelet-activating platelet G-protein factorreceptor factor receptor activating coupled factor receptor; receptorPAF activity receptor; platelet- activating factor receptor Immunesystem gene suppressed by the peptide 1 NM_006864 0.662 leukocyteleukocyte receptor immunoglobulin-like immunoglobulin-like activityreceptor, subfamily B receptor, subfamily (with TM and ITIM B (with TMand ITIM domains), member 3 domains), member 3 2 NM_007052 0.662 NADPHoxidase 1 NADPH oxidase 1 superoxide- isoform long; NADPH generatingoxidase 1 isoform NADPH oxidase short; NADPH activity; oxidase 1 isoformoxidoreductase long variant activity; voltage-gated proton channelactivity 3 NM_002258 0.653 killer cell lectin- killer cell lectin- sugarbinding; like receptor like receptor transmembrane subfamily B, member 1subfamily B, member 1 receptor activity 4 NM_001776 0.646 ectonucleosideectonucleoside apyrase triphosphate triphosphate activity;diphosphohydrolase 1 diphosphohydrolase 1 magnesium ion binding;hydrolase activity 5 NM_016523 0.644 killer cell lectin- killer celllectin- transmembrane like receptor like receptor receptor subfamily F,member 1 subfamily F, member 1 activity 6 NM_014442 0.641 sialic acidbinding sialic acid binding sugar binding; Ig-like lectin 8 Ig-likelectin 8 transmembrane receptor activity; cell adhesion moleculeactivity 7 NM_004133 0.635 hepatocyte nuclear hepatocyte nuclear steroidfactor 4, gamma factor 4, gamma hormone receptor activity; steroidbinding; transcription factor activity 8 NM_002121 0.633 major majorBeryllium class II major histocompatibility histocompatibility disease,histocompatibility complex, class II, DP complex, class II, chronic,complex beta 1 DP beta 1 precursor susceptibility antigen to 9 NM_0003970.631 cytochrome b-245, cytochrome b-245, Chronic voltage-gated betapolypeptide beta polypeptide granulomatous ion channel (chronic (chronicdisease, activity; granulomatous granulomatous X-linked electrondisease) disease) transporter activity; oxidoreductase activity 10NM_004750 0.626 cytokine receptor- cytokine receptor- Cold- receptorlike factor 1 like factor 1 induced activity sweating syndrome 11NM_003891 0.618 protein Z, vitamin K- protein Z, vitamin trypsindependent plasma K-dependent plasma activity; glycoprotein glycoproteinprotein binding; calcium ion binding; chymotrypsin activity 12 NM_0022600.603 killer cell lectin- killer cell lectin- sugar binding; likereceptor like receptor transmembrane subfamily C, member 2 subfamily C,member 2 receptor activity 13 NM_005545 0.579 immunoglobulinimmunoglobulin protein superfamily superfamily binding containingleucine- containing leucine- rich repeat rich repeat 14 NM_001311 0.561cysteine-rich protein cysteine-rich zinc ion 1 (intestinal) protein 1binding (intestinal) 15 NM_004528 0.561 microsomal microsomal peroxidaseglutathione S- glutathione S- activity; transferase 3 transferase 3glutathione transferase activity 16 NM_018661 0.528 defensin, beta 103defensin, beta 103, antimicrobial precursor peptide activity;Gram-positive antibacterial peptide activity 17 NM_002124 0.502 majormajor Pemphigoid, MHC class II histocompatibility histocompatibilitysusceptibility receptor complex, class II, DR complex, class II, toactivity beta 1 DR beta 1 precursor 18 Y00815 0.475 protein tyrosineprotein tyrosine antigen; phosphatase, receptor phosphatase, cell type,F receptor type, F surface isoform 1 glycoprotein; precursor; proteinglycoprotein; tyrosine immunoglobulin phosphatase, superfamily; receptortype, F LAR isoform 2 precursor gene; leukocyte common antigen; neuralcell adhesion molecule; transmembrane protein 19 NM_004636 0.459 semadomain, sema domain, immunoglobulin domain immunoglobulin (Ig), shortbasic domain (Ig), short domain, secreted, basic domain, (semaphorin) 3Bsecreted, (semaphorin) 3B 20 NM_001783 0.4 CD79A antigen CD79A antigentransmembrane (immunoglobulin- isoform 1 receptor associated alpha)precursor; CD79A activity antigen isoform 2 precursor 21 NM_004106 0.382Fc fragment of IgE, Fc fragment of IgE, receptor high affinity I, highaffinity I, signaling receptor for; gamma receptor for; gamma proteinpolypeptide polypeptide activity; precursor transmembrane receptoractivity; IgE binding 22 U77604 0.349 microsomal microsomal glutathioneglutathione S- glutathione S- transferase transferase 2 transferase 2activity; enzyme activator activity 23 NM_002118 0.335 major majorchaperone histocompatibility histocompatibility activity; MHC complex,class II, DM complex, class II, class II beta DM beta precursor receptoractivity 24 NM_002123 0.328 major major Creutzfel histocompatibilityhistocompatibility dt-Jakob complex, class II, DQ complex, class II,disease, beta 1 DQ beta 1 precursor variant, resistance to 25 K011710.319 major major MHC class II antigen; histocompatibilityhistocompatibility receptor class II complex, class II, DR complex,class II, activity antigen; alpha DR alpha precursor histocompatibilityantigen; major histocompatibility complex 26 NM_006120 0.298 major majorhistocompatibility histocompatibility complex, class II, DM complex,class II, alpha DM alpha precursor

The present inventor has verified the validity of this diagnosis methodusing 55000 cancer-related genes. When such a great number ofcancer-related genes are used, DNA chip technology is preferably used.In the DNA chip technology, each DNA of the cancer-related genes isimmobilized so as to be arrayed on a slide; the above-mentioned labeledcRNA solution is dropped into the slide; the cRNA which is nothybridized is removed by washing; and fluorescence intensity of eachgene position is detected by a scanner. The results are compared withthe result which is obtained by conducting the same process using theabove-mentioned control solution. Thus, cancer-related genes of whichexpression is increased or decreased when cells or tissues are treatedwith the peptide of the present invention can be exhaustively analyzed.

Therefore, a combination of such a cancer-related gene, the peptide ofthe present invention, and a polynucleotide encoding the peptide isuseful as a cancer diagnostic reagent kit for determining a tendency ofcanceration or malignancy of cancer.

[Diagnosis Method 3]

The peptide of the present invention is also used as an antigen forpreparing a specific antibody against the peptide. The specific antibodyis useful as a diagnostic reagent. The antibody may be either polyclonalor monoclonal antibody, and these antibodies can be prepared by knownmethods. For example, the polyclonal antibody can be prepared as anantiserum by immunizing an animal such as mouse, rat, or rabbit with thepeptide of the present invention. The antiserum may be further purified.The monoclonal antibody can be prepared by a hybridoma method:extracting spleen cells from the above-mentioned sensitized animal andfusing the spleen cells with myeloma cells to form hybridomas.

In the immunization, an appropriate adjuvant may be used. In addition,the peptide of the present invention may be used as a conjugate with acarrier protein such as keyhole limpet hemocyanin for increasing theimmunogenicity of the peptide.

The antibody against the peptide of the present invention is used as adiagnostic reagent for determining malignancy of cancer, wherein aspecimen, such as cancer tissues or cells, is stained by using thespecific antibody labeled with an appropriate fluorescence dye or thelike. When the peptide of the present invention is detected in thetissues or cells, it is suggested that cancer is advancing. The amountof the peptide can be determined by measuring the fluorescenceintensity. A large amount of the peptide indicates that the malignancyof cancer is considerably high.

Additionally, the antibody against the peptide of the present inventioncan be used as a diagnostic reagent for measuring a tendency ofcanceration (easiness of conversion to cancer: a degree of risk ofcanceration). In such a use of the antibody, a specimen of the tissuesor cells shown in diagnosis methods 1 and 2 is stained using the labeledantibody. When the peptide of the present invention is detected in thesetissues or cells, it is suggested that the tissues or cells are exposedto cancer-inducing stress, i.e., a degree of risk of canceration ishigh. When the amount of the peptide of the present invention is large,it is suggested that the tendency is further higher. When the specimenis cancer cells, a large amount of the peptide suggests that themalignancy of the cancer is high.

Furthermore, the antibody against the peptide of the present inventioncan be expected to have an anticancer effect such as suppression ofcancer cell proliferation as a cancer-cell proliferation-suppressingdrug. In addition, the antibody can be expected to block malignantalteration or mutation acceleration to which normal cells are subjectedas a cancer-preventing drug. Furthermore, by binding the antibody to ananticancer agent, a drug which may be used for missile therapy can beobtained.

EXAMPLES

Examples of the present invention will now be described, but the presentinvention is not limited to these examples.

Example 1 Preparation and Sequencing of the Peptide of the PresentInvention

(Cell: UTC-8)

The squamous-cell carcinoma cell (UTC-8: FERM BP-08611) is a highlydifferentiated type with high metastasis potential and keratinizationtendency. The cell also has high adhesion and proliferation ability andbecomes confluent in 5 to 7 days after inoculating 1×10⁵ cells into aculture medium (5 ml) in a T25 culture flask (Falcon).

(Cell-Culturing Condition)

The following processes were performed: inoculating 1×10⁶ UTC-8 cellsinto 10 T150 culture flasks (Falcon); performing initial culture in 20ml RPMI1640 culture medium with 10% FCS (GIBCO); and additionallysupplying 30 ml of the culture medium when the proliferation reachedabout 30% of the culture flask dimension. The culture was performed in5% CO₂ and 95% humidity at an incubator internal temperature of 37° C.When the cells become 80% confluent (the proliferation of the cells ismaintained at the point just before the cell proliferation reaches itspeak), the antigen peptide bound to HLA was eluted from the cellmembrane surfaces.

(Antigen Peptide-Eluting Procedure)

The culture medium for the UTC-8 cells was removed when the cells became80% confluent in each T150 culture flask (Falcon). The cells were washedwith Hanks solution once and then with 30 ml of a PBS solution notcontaining divalent calcium ion and divalent magnesium ion twice. Then,after the sufficient removal of the solution, 10 ml of acitrate-phosphate buffer having a pH of 3.3 to 3.4 was added to eachflask, and the cells were left at a room temperature for 2 min.

Then, the citrate-phosphate buffer solution was collected andcentrifuged at 1200 rpm for 7 min, and the supernatant was filteredthrough a 0.45 μm filter (Millex-HV PVDF: MILLIPORE). The filtrate wasfurther filtered through a 0.22 μm filter. The filtrate was desaltedwith Sep-Pac C18 cartridge (Waters), and the target substance bound tothe cartridge was eluted with a 60% (v/v) acetonitrile aqueous solutionas a crude extract solution. The thus obtained crude extract solutionwas frozen and stored at −20° C. for later HPLC.

The cells received the above-described treatment were washed,immediately after the collection with the citrate-phosphate buffersolution having a pH of 3.3 to 3.4, with 30 ml of Hanks solution twice.Then, the cells were recultured in 30 ml of RPMI 164 medium containing10% FCS. This treatment was subjected to the same cells once a day forsuccessive 4 days.

(Lyophilization and Redissolution of Crude Extraction)

The crude extract solution obtained in above was lyophilized using alyophilizer (FD-1000: EYELA) under conditions at a trapping temperatureof −40° C. and at a degree of vacuum of 15 Pa or less. The dried samplewas redissolved in 5 ml of the citrate-phosphate buffer solution havinga pH of 3.3 to 3.4. At this stage, the total protein amount determinedby Lowry method was 400 μg.

(Two-Dimensional Electrophoresis)

Ten micrograms of the thus obtained molecule was applied totwo-dimensional electrophoresis, but no band was visible to the nakedeye by Coomassie Brilliant Blue (CBB) R250 staining (detectionsensitivity: 1 μg) and also by silver staining having a sensitivity ashigh as about 1000 times that of the CBB staining, (theoreticalsensitivity: 1 ng, actual sensitivity: about 20 times that of the CBBstaining). This suggests that the peptide molecule physically passesthrough a gel (silica gel C18) generally used in the two-dimensionalelectrophoresis. This is an unusual characteristic of the peptidemolecule of the present invention.

(Fractionating by Gel Filtration)

Since the molecule of the present invention cannot be detected by usualtwo-dimensional electrophoresis, it was tried to detect the molecule byan HPLC system. The target peptide was obtained as a fraction obtainedby using an AKTA Explorer 10 (Amersham Pharmacia Biotech) HPLC systemand Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech) as agel-filtration column; using a citrate-phosphate buffer solution (0.1 Mcitric acid and 0.2 M phosphoric acid) having a pH of 6.8 as an elutionbuffer at a flow rate of 0.2 ml/min; and isolating a peak in a fractionat an elution volume of 20.8 to 22.8 ml by using a detection wavelengthof 214 nm. The obtained fraction was lyophilized and then redissolved insterilized redistilled water for desalting. The desalting was performedby fractionating by the AKTA Explorer 10 (Amersham Pharmacia Biotech)HPLC system and Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech)as a gel-filtration column using distilled water as a solvent at a flowrate of 0.2 ml/min. The target peptide was obtained as a fraction at anelution volume of 11.4 to 20.0 ml.

FIG. 1 shows an elution profile of the gel filtration.

The peptide was lyophilized using the lyophilizer FD-1000 (EYELA) andstored at −20° C. for later mass spectrometry.

(Optimal pH)

The optimal pH of the elution buffer was 6.8 to 7.2. When the pH of theelution buffer was 7.3 or more, peaks were further divided into smallerpeaks. When the pH of the elution buffer was 6.7 or less, peaks, whichwere isolated from each other at a pH of 6.8, lapped over each other,and the gel filtration resolution was decreased.

(Detection Wavelength)

The peptide is characterized by the fact that the peak of the peptide inthe crude extract solution can be separately detected by usingphysiological activity as an indicator only when a detection wavelengthof 214 nm is used. However, the peptide cannot be detected when adetection wavelength of 254 to 257 nm or of 280 nm, which are generallyused in peptide detection, is used. These results suggest that the aminoacid composition of the peptide has extremely low contents of tyrosine(absorption wavelength: 280 nm), tryptophan (absorption wavelength: 280nm), and phenylalanine (absorption wavelength: 257 nm); or that thepeptide does not substantially contain such amino acids. The use of adetection wavelength of 214 nm has a demerit such that many interferingsubstances are also detected by ultraviolet absorption spectrometry.However, it can precisely measure a protein amount (quantitativedetection range: 5 to 1000 μg) regardless of types of proteins. The 214nm is the most appropriate wavelength for, as in this experiment,precisely measuring a concentration of unknown peptide in a crudeextract solution by detecting a peak utilizing peptide-bond absorptionwithout denaturation of the peptide to maintain the physiologicalactivity. On the other hand, the quantitative detection property wasfurther improved by using a detection wavelength of 205 nm when thecrude extract solution containing the peptide was applied to a desaltingcolumn (Sep-Pack 18) or to a molecular sieve (Centricon YM-3) forremoving foreign substances such as nucleic acids as far as possible, byintending only purification of the peptide without any concern formaintaining the physiological activity. Therefore, when a peak in thecrude extract solution was detected using the physiological activity asan indicator, a detection wavelength of 214 nm was used, and when theprecise quantitative determination was necessary, a detection wavelengthof 205 nm was used.

(Fractionating Flow Rate in Column)

The fractionating flow rate is important for separating the peptide.Namely, the flow rate of an elution buffer is determined so thatmolecules slowly pass through the column to effectively utilizeadvantages of the molecular sieve. When the flow rate was 0.28 ml/min,adjacent peaks lapped over each other; thus, the separation resolutionwas insufficient. For the detection of the peak of the presentinvention, a low flow rate of 0.14 ml/min was optimal. Each fraction wasdetermined to be 0.1 ml/well from the view point of the width of thepeak, and was collected in a 96-well plate (Nunc).

(Structure Analysis of the Peptide by Mass Spectrometer)

The peptide fraction that was stored after lyophilization wasredissolved in sterilized redistilled water, and 5 μg as a protein wasseparated by ProteinChip Series 4000 system (Ciphergen). The sample (5μg) was bound to the ProteinChip by using MilliQ water as a binding/washbuffer in normal-phase ProteinChip and using 100 mM sodium acetate (pH4.0) as a binding/wash buffer in cation exchange ProteinChip, and wasmeasured by using alpha-cyano-4-hydroxy-cinnamic acid (CHCA) as anenergy-absorbing molecular in a mass range of 800 to 2500 m/z. As aresult, a peak of a molecular weight which was thought to be that of thetarget peptide was detected. By using the detection conditions clarifiedby the above-described processes as a reference, the target peptidetrapped on the chip was analyzed by a mass spectrometer, QSTAR XLLC/MC/MS system (Applied Biosystems). Then, on the basis of the resultsof time-of-flight (TOF mass spectrometry) analysis of the fragmentedpeptide-constituting portion, a data base (Mascot Search Results: MatrixScience) was searched for a sequence of the peptide to reveal that thepeptide had the following peptide sequence of which function was unknownas of Jan. 27, 2005.

Peptide: (SEQ ID NO: 1) Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu-Ser-Lys

Example 2 Confirmation of Expression of the Peptide of the PresentInvention in Original Cancer Cell UTC-8

The fact that the peptide obtained in Example 1 was actually expressedin the original cancer cell UTC-8 at a gene level was confirmed asfollows:

UTC-8 cells after the extraction of the peptide of the present inventionwere recultured, and total RNA was extracted from the recultured UTC-8cells after 2 hr, 4 hr, and 6 hr from the extraction, respectively, byusing an RNA extraction kit (QIAGEN). Then, cDNA was synthesized usingeach of the extracted total RNA as a template to produce a cDNA pool. Onthe basis of the amino acid sequence (SEQ ID NO: 1) of the peptide, thefollowing primers were designed. Fw primers (DNAs encoding fourcontiguous amino acids of the peptide were synthesized so that theN-terminal of the four contiguous amino acids shifted one by one fromthe N-terminal of the peptide toward the C-terminal.)

MHC 1-4 CAR CCN CAR TTY (SEQ ID NO: 2) MHC 2-5 CCN CAR TTY GGN (SEQ IDNO: 3) MHC 3-6 CAR TTY GGN AGR (SEQ ID NO: 4) MHC 4-7 TTY GGN AGR AGR(SEQ ID NO: 5) MHC 5-8 GGN AGR AGR ATG (SEQ ID NO: 6) MHC 6-9 AGR AGRATG GAR (SEQ ID NO: 7) MHC 7-10 AGR ATG GAR TCN (SEQ ID NO: 8) MHC 8-11ATG GAR TCN AAR (SEQ ID NO: 9) (N: A, T, C, or G, R: G or A, Y: C or T)Rv primer (a gene sequence produced for an amino acid sequence at theC-terminal of the peptide): Oligo-dT primer (poly-A primer)

Here, the poly-A primer was used as the Rv primer in order to obtaincDNA synthesized from mRNA.

Complementary DNAs were synthesized by reverse transcription from totalRNA which was extracted at each time to produce a cDNA pool. Then, PCRwas performed using this cDNA pool as templates and using primerssynthesized as described above under conditions at 94° C. for 3 min, at55° C. for 1 min, and at 74° C. for 1 min as one cycle. The cycle wasrepeated 35 cycles in total. After agarose-gel electrophoresis, 12 bandswere selected from a sample of which RNA was extracted after 4 hr whenthe signal intensity was largest. The size of each band was about 500 to2000 bp. Genes of these bands extracted from the gel were inserted intopGM easy vectors.

Insert of the genes into 12 types of the pGM easy vectors was checked,and 8 samples per one type of the vector were sequenced. As a result, itwas confirmed that all the sequences of the 8 samples included a commonsequence shown in Table 3 below, though the DNA lengths of the sequenceswere different from those of each other.

TABLE 3

The result shows the fact that a gene being common in or complementaryto the gene encoding the peptide of the present invention is surelypresent in original cancer cell UTC-8 and that the peptide is derivedfrom UTC-8 cells.

Example 3 Artificial Synthesis of the Peptide of the Present Inventionby Fmoc Method

The peptide was artificially synthesized by an in vitro synthesis system(Abacus: Sigma Genosys).

The peptide was synthesized using an activated cellulose membrane(SPOTs: Sigma Genosys) as follows: The α-amino groups of amino acidswere protected with 9-fluorenyl methoxy carbonyl (Fmoc) and the carboxylgroups were protected with an active ester (Opfp or Odhbt). These aminoacid derivatives were dissolved in 1-methyl-2-pyrrolidinone (NMP). About1 μl of the amino acid derivative solution (equivalent to 100 to 200 μgof Fmoc-amino acid) was spotted on the membrane at each marked positionwhere a spacer arm was bound thereto. The end of the spacer arm had afree amino group, and the spots were colored to blue with bromophenolblue (BPB). An amido bond was formed by the reaction between the aminogroup on the membrane and the active ester. Then, capping was carriedout as follows: Excess amino acid derivatives were rinsed away withdimethylformamide (DMF), and then unreacted amino group was acetylatedby treating with acetic anhydride/DMF so as to be lost the reactivity.Then, the Fmoc group protecting the amino group was removed by secondaryamine, piperidine/DMF, in order to let the amino group free for thesubsequent extension reaction. Additionally, after all synthesisprocesses were completed, deprotection of the side chains was carriedout. Namely, a t-butyl alcohol-based protecting group (Pmc, OtBu, Trt,tBoc, tBu, etc.) protecting the reactive side chains was removed byusing trifluoroacetate (TFA) mixed with dichloromethane(DCM)/triisobutylsilane. Thus, the target peptide was artificiallysynthesized.

Example 4 Influence of the Artificially Synthesized Peptide on NormalCell Gene

(a) The peptide obtained in Example 3 was added to a human normalperipheral blood monocyte culture system (10 to 100 μg peptide/1×10⁶ to1×10⁷ monocytes), and they were cocultured for 7 hr. Then, mRNA wasextracted from the monocytes and was applied to analysis forup-regulation and down-regulation in respect to about 55000 human genesby using DNA chip system (Clontech). Table 4 shows a part of the genes.

TABLE 4 A. Enhanced Gene Expression Expression Amount Amount beforeafter Treatment Treatment with with Gene Gene the the Enhancement No.Gene Name Bank Peptide Peptide Ratio Description 1. Onco- gene 1 21713NM_004339.2 GE480864 DISCOVERY 0.168670654 7.741943359 45.89976479zn87b11y5 Stratagene lung carcinoma 937218 cDNA clone IMAGE: 565149 5′similar to contains Alu repetitive element; contains element MER22repetitive element; 2 32050 CB999164.1 GE540894 DISCOVERY 0.3448181159.805541992 28.43685282 disrupted in renal carcinoma 1 (DIRC1) 3 34854NM_198502.1 GE603511 DISCOVERY 0.040802002 1.098205566 26.91548242 Homosapiens NEUROBLASTOMA COT 25-NORMALIZED cDNA clone CS0DC018YE24 3-PRIME4 46997 AL831827.1 GE547122 DISCOVERY 11.17044067 236.181823721.14346521 clone N11 NTera2D1 teratocarcinoma mRNA 5 37781 AI478531.1GE645872 DISCOVERY 2.533325195 32.40939331 12.79322267 NEUROBLASTOMA COT25-NORMALIZED cDNA clone CS0DC011YD03 5-PRIME 6 7394 AA021565.1 GE707587DISCOVERY 29.597229 350.270813 11.8345813 HELA CELLS COT 25-NORMALIZEDcDNA clone CS0DK007YM05 3-PRIME 7 22388 CD244420.1 GE692174 DISCOVERY60.32098389 571.2325439 9.469881078 zb91f05s1Soares_parathyroid_tumor_(—) NbHPA cDNA clone IMAGE: 320193 3′ 8 6231BF973345.1 GE54360 DISCOVERY 0.583343506 5.453491211 9.348679048 breastcarcinoma amplified sequence 1 (BCAS1) 9 34979 BC041456.1 GE631189DISCOVERY 2.651672363 24.171875 9.115709518 yu38e06r1 Soares ovary tumorNbHOT cDNA clone IMAGE: 236098 5′ 10 4210 NM_152577.1 GE493259 DISCOVERY61.90475464 393.7416382 6.360442594 RAS-like, family 11, member A(RASL11A) 11 36631 AW302705.1 GE53805 DISCOVERY 71.11248779 417.55.87098009 RAB43, member RAS oncogene family (RAB43), mRNA 12 29317AA601191.1 GE551636 DISCOVERY 2.891571045 10.05682373 3.477979124 T-cellleukemia/lymphoma 6 (TCL6), transcript variant TCL6a1 13 14195NM_002170.2 GE55306 DISCOVERY 2.811950684 7.21875 2.567168067 B-celltranslocation gene 4 (BTG4) 14 2183 AA861218.1 GE80525 DISCOVERY3.408691406 8.622650146 2.529607148 neuro-oncological ventral antigen 1(NOVA1), transcript variant 1 15 22675 BX117842.1 GE516913 DISCOVERY81.83721924 204.4117737 2.497784939 endogenous retroviral family W,env(C7), member 1 (syncytin) (ERVWE1), mRNA 16 3858 AI283196.1 GE59314DISCOVERY 9.184204102 22.61538696 2.46242208 preferentially expressedantigen in melanoma (PRAME), transcript variant 3 17 8431 NM_021081.3GE56485 DISCOVERY 7.641021729 18.609375 2.435456365 Ras-associatedprotein Rap1 (RBJ) 18 18829 NM_002958.1 GE81500 DISCOVERY 6.88333129915.3157959 2.225055752 v-myc myelocytomatosis viral related oncogene,neuroblastoma derived (avian) (MYCN) 19 8440 NM_016232.4 GE79419DISCOVERY 7.225799561 15.71429443 2.174748179 mab-21-like 2 (C elegans)(MAB21L2) 20 3642 AW594132.1 GE543964 DISCOVERY 1627.052002 3306.9428712.032475217 RAB2, member RAS oncogene family (RAB2) 21 38566 NM_031273.1GE84875 DISCOVERY 7.339630127 14.54998779 1.982387061 RAS proteinactivator like 2, mRNA (cDNA clone IMAGE: 5399841), with apparentretained intron 22 24910 NM_018556.2 GE60436 DISCOVERY 29.0133361855.05334473 1.897518589 platelet-derived growth factor beta polypeptide(simian sarcoma viral (v-sis) oncogene homolog) (PDGFB), transcriptvariant 2 23 6329 BX116538.1 GE58014 DISCOVERY 37.80822754 67.536590581.786293486 mutated in colorectal cancers (MCC) 2. Immune System Gene 125767 INCYTE GE59636 DISCOVERY 130.6451721 4927.383301 37.71577029 tumornecrosis factor (TNF UNIQUE superfamily, member 2) (TNF) 2 29703BX103139.1 GE59980 DISCOVERY 0.660003662 19.8302002 30.04559116chemokine (C—X—C motif) ligand 9 (CXCL9) 3 28615 AA504638.1 GE520093DISCOVERY 2.739135742 20.69332886 7.554692722 IL3-UT0117-080301-496-D07UT0117 Homo sapiens cDNA 4 30707 AI809890.1 GE54001 DISCOVERY2.853668213 21.05661011 7.378787069 immunoglobulin superfamily, member4C (IGSF4C) 5 21607 NM_002960.1 GE80951 DISCOVERY 3.44628906324.92727661 7.233077713 colony stimulating factor 1 (macrophage) (CSF1),transcript variant 1 6 51904 H08511.1 GE893705 DISCOVERY 2.30487060511.3684082 4.932341181 interleukin 1 family, member 8 (eta) (IL1F8),transcript variant 1 7 22254 NM_176891.2 GE80659 DISCOVERY 26.98571777122.6947327 4.546654408 interleukin 4 (IL4), transcript variant 1 845170 BM671892.1 GE53155 DISCOVERY 38.31506348 85.25881958 2.225203663transforming growth factor beta 1 induced transcript 1 (TGFB1I1) 9 22100BX457477.2 GE616415 DISCOVERY 328.440918 608.4909058 1.852664734IL5-CI0001-181000-178-f03 CI0001 Homo sapiens cDNA B. Suppressed GeneExpression Expression Amount Amount before after Treatment Treatmentwith with Gene Gene the the Suppression No. Gene Name Bank PeptidePeptide Ratio Description 1. Cancer Sup- pressing Gene 1 35687BX108016.1 GE82100 DISCOVERY 103.9224243 0.225219727 461.4268293 heatshock protein 75 (TRAP1) 2 36334 BF433017.1 GE82101 DISCOVERY 341.4409182.079650879 164.1818449 heat shock 70 kDa protein 14 (HSPA14) 3 37600AW979088.1 GE62125 DISCOVERY 982.9432373 8.42855835 116.6205651 AHA1,activator of heat shock 90 kDa protein ATPase homolog 1 (yeast) (AHSA1)4 30909 NM_018412.2 GE79080 DISCOVERY 987.7108154 11.4736938586.08481528 tumor rejection antigen (gp96) 1 (TRA1) 5 8280 BC063301.1GE62103 DISCOVERY 758.4154053 9 84.26837836 brain specific protein(CGI-38) 6 11438 NM_152271.2 GE82058 DISCOVERY 1207.091431 14.7441711481.86905991 serologically defined breast cancer antigen 84 (SDBCAG84),transcript variant 1 7 11261 NM_018559.2 GE56225 DISCOVERY 379.13385016.257141113 60.59218471 breast cancer metastasis suppressor 1 (BRMS1) 850395 NM_004480.3 GE79552 DISCOVERY 470.2086792 8.763153076 53.65747638Ras suppressor protein 1 (RSU1), transcript variant 1 9 52111 AK056875.1GE61359 DISCOVERY 593.6870728 13.6000061 43.65344164 leukemia cellnormalized cDNA library cDNA clone LEU1757_26_C2 2. Immune System Gene 120881 NM_001482.1 GE61110 DISCOVERY 6599.050293 38.16882324 172.891112natural killer cell transcript 4 (NK4) 2 47973 NM_004394.1 GE81458DISCOVERY 1480.68396 9.638885498 153.615681 CD8 antigen, betapolypeptide 1 (p37) (CD8B1), transcript variant 5 3 10791 W00901.1GE80378 DISCOVERY 517.4453125 3.42855835 150.9221252 HLA-B associatedtranscript 5 (BAT5) 4 15907 BU899259.1 GE80960 DISCOVERY 912.86413576.363647461 143.4498283 intercellular adhesion molecule 2 (ICAM2) 516756 AI707455.1 GE81522 DISCOVERY 395.0359497 2.80645752 140.7596399major histocompatibility complex, class I, E (HLA-E) 6 34736 AV736303.1GE80100 DISCOVERY 3974.221191 29.0786438 136.6714768 adhesion moleculeAMICA (AMICA) 7 39991 AI252940.1 GE61199 DISCOVERY 2414.74877917.77420044 135.8569567 lymphotoxin beta (TNF superfamily, member 3)(LTB), transcript variant 2 8 41726 AI453596.1 GE58507 DISCOVERY4512.05127 33.50683594 134.660619 apoptosis-associated speck- likeprotein containing a CARD (ASC), transcript variant 1 9 45767 BG190549.1GE79374 DISCOVERY 2359.263184 18.125 130.1662446 HLA-B associatedtranscript 1 (BAT1), transcript variant 1 10 42614 AW296107.1 GE81744DISCOVERY 626.2438965 4.848480225 129.1629268 killer cell lectin-likereceptor subfamily K, member 1 (KLRK1) 11 20551 AK024566.1 GE86210DISCOVERY 2811.726807 22.93103027 122.6166802 IL2-inducible T-cellkinase (ITK) 12 38103 NM_173695.1 GE61298 DISCOVERY 703.88751225.777770996 121.8268278 tumor necrosis factor receptor superfamily,member 7 (TNFRSF7) 13 37068 AW971488.1 GE58019 DISCOVERY 6960.16113357.38461304 121.2896762 immediate early response 2 (IER2) 14 4899BX115183.1 GE80961 DISCOVERY 536.4679565 4.5 119.2151015 interferon(alpha, beta and omega) receptor 2 (IFNAR2), transcript variant 1 1543032 BC013284.2 GE58028 DISCOVERY 519.3710938 4.445770264 116.8236465interferon, gamma-inducible protein 16 (IFI16) 16 42813 NM_001763.1GE58393 DISCOVERY 4447.713379 38.51351929 115.4844704 granzyme A(granzyme 1, cytotoxic T-lymphocyte- associated serine esterase 3)(GZMA) 17 22326 NM_014396.2 GE82907 DISCOVERY 1102.541626 9.742858887113.1640763 immune associated nucleotide 2 (hIAN2) 18 37445 NM_007068.2GE60016 DISCOVERY 1671.436768 14.875 112.365497 mal, T-celldifferentiation protein (MAL), transcript variant d 19 22890 BX119833.1GE86305 DISCOVERY 1112.63855 9.902160645 112.3632094 NK inhibitoryreceptor precursor (NKIR) 20 16478 NM_020530.3 GE54528 DISCOVERY663.3167725 6.018188477 110.2186771 tumor necrosis factor (ligand)superfamily, member 12 (TNFSF12), transcript variant 1, mRNA 21 52539AI133415.1 GE82473 DISCOVERY 17346.24609 158.6896667 109.3092351 majorhistocompatibility complex, class II, DR alpha (HLA-DRA) 22 41803NM_178313.1 GE735470 DISCOVERY 89.23596191 0.820007324 108.8233718programmed cell death 2 (PDCD2), transcript variant 2 23 41817NM_016292.1 GE79992 DISCOVERY 10618.77539 98.42697144 107.8848128 majorhistocompatibility complex, class I, F (HLA-F) 24 18884 NM_015604.2GE57715 DISCOVERY 339.244873 3.387084961 100.1583595 (clone 38-1) MHCclass I mRNA fragment. 25 8922 NM_024077.2 GE57504 DISCOVERY 10964.4248110.8526306 98.90991981 interferon induced transmembrane protein 1(9-27) (IFITM1) 26 24969 AV752332.1 GE60370 DISCOVERY 2446.13867225.71212769 95.13559911 CD69 antigen (p60, early T-cell activationantigen) (CD69) 27 8634 BC043004.2 GE566269 DISCOVERY 947.687510.19564819 92.9501962 perform 1 (pore forming protein) (PRF1) 28 38879NM_182832.1 GE476899 DISCOVERY 2933.067871 31.93505859 91.84476247interferon stimulated gene 20 kDa (ISG20) 29 49586 BG289120.1 GE58687DISCOVERY 248.4822998 2.715911865 91.49129726 interleukin-1 receptor-associated kinase 4 (IRAK4) 30 11651 AW850450.1 GE85351 DISCOVERY1144.841431 12.83999634 89.16213062 implantation-associated protein(DKFZp564K142) 31 53172 NM_022749.4 GE59644 DISCOVERY 577.42260746.560333252 88.01726761 transforming growth factor, beta 1(Camurati-Engelmann disease) (TGFB1) 32 49622 CB052158.1 GE59962DISCOVERY 3421.524414 39.68115234 86.22542976 intercellular adhesionmolecule 3 (ICAM3) 33 22868 NM_152285.1 GE79323 DISCOVERY 539.82910166.525421143 82.72708991 lymphocyte cytosolic protein 2 (SH2 domaincontaining leukocyte protein of 76 kDa) (LCP2) 34 23827 BG221408.1GE59882 DISCOVERY 2403.98877 29.7802124 80.72436613 majorhistocompatibility complex, class II, DM alpha (HLA-DMA) 35 18139AA938869.1 GE890404 DISCOVERY 2660.284912 33.11999512 80.32262392 majorhistocompatibility complex, class II, DQ alpha 1 (HLA-DQA1) 36 22789BX104097.1 GE61439 DISCOVERY 731.80896 9.725494385 75.24645339 CASP8 andFADD-like apoptosis regulator (CFLAR) 37 11236 NM_003937.1 GE58794DISCOVERY 4738.49707 65.4675293 72.37934777 natural killer cell group 7sequence (NKG7), mRNA 38 10204 AW197778.1 GE81039 DISCOVERY 6969.714294434 71.64699451 interferon-related developmental regulator 1(IFRD1) 39 49170 AA577911.1 GE61247 DISCOVERY 713.4000244 10.0909118770.69728028 interferon gamma receptor 2 (interferon gamma transducer 1)(IFNGR2) 40 42474 AW137161.1 GE62833 DISCOVERY 1164.558472 17.0377502468.35165764 programmed cell death 6 (PDCD6) 41 52342 NM_006147.2 GE54992DISCOVERY 1200.007446 18.71737671 64.11194608 apoptosis-related proteinPNAS-1 (FLJ39616) 42 42573 NM_022648.2 GE79383 DISCOVERY 953.407836915.75 60.53383092 T-cell activation protein (PGR1) 43 24440 NM_182936.1GE592149 DISCOVERY 14 0.234771729 59.63239308 THYMUS cDNA cloneCS0CAP007YL06 5-PRIME 45 24440 NM_182936.1 GE592149 DISCOVERY 140.234771729 59.63239308 THYMUS cDNA clone CS0CAP007YL06 5-PRIME 46 24440NM_182936.1 GE592149 DISCOVERY 14 0.234771729 59.63239308 THYMUS cDNAclone CS0CAP007YL06 5-PRIME 47 46186 NM_032746.1 GE82057 DISCOVERY1407.181763 25.55319214 55.06872703 cell death-regulatory protein GRIM19(GRIM19) 48 39926 INCYTE GE82589 DISCOVERY 17409.49609 317.710937554.79665331 thymosin-like 6 (TMSL6) UNIQUE 49 47689 AK097380.1 GE61992DISCOVERY 3803.877441 72.08435059 52.7698094 killer cell lectin-likereceptor subfamily B, member 1 (KLRB1) 50 12812 CF135919.1 GE55506DISCOVERY 1176.72522 24.53225708 47.96644744 ovarian carcinomaimmunoreactive antigen (OCIA) 51 15994 R25284.1 GE79364 DISCOVERY11397.33301 239.4736938 47.5932568 beta-2-microglobulin (B2M) 52 52071BM698907.1 GE86827 DISCOVERY 415.7030029 9.42855835 44.08977359interferon (alpha, beta and omega) receptor 1 (IFNAR1) 53 40727BG573885.1 GE55241 DISCOVERY 1204.090942 28.16665649 42.74880629 linkerfor activation of T cells (LAT) 54 11635 AI376607.1 GE80662 DISCOVERY106.1363525 2.5 42.45454102 thymosin, beta 4, Y-linked (TMSB4Y) 55 4403N26032.1 GE56202 DISCOVERY 891.694458 21.96551514 40.595199 programmedcell death 4 (neoplastic transformation inhibitor) (PDCD4), transcriptvariant 2 56 14949 BX110547.1 GE81738 DISCOVERY 1020.042969 25.2676086440.36958872 programmed cell death 10 (PDCD10), transcript variant 1 5751004 BM670853.1 GE59115 DISCOVERY 345.9705811 8.725006104 39.65276092tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10) 58 17177AI274757.1 GE54607 DISCOVERY 1033.54187 26.16049194 39.50773832 tumornecrosis factor superfamily, member 5- induced protein 1 (TNFSF5IP1) 599938 NM_152288.1 GE87537 DISCOVERY 11.78378296 0.299987793 39.28087487interleukin 17D (IL17D) 60 21487 NM_006861.4 GE58813 DISCOVERY387.6266479 9.909088135 39.11829653 programmed cell death 2 (PDCD2),transcript variant 1 61 37834 BU608350.1 GE57222 DISCOVERY 545.760253914.10638428 38.68888322 proteasome (prosome, macropain) 26S subunit,non-ATPase, 1 (PSMD1) 62 42661 C04533.1 GE60353 DISCOVERY 1506.33935539.14474487 38.4812664 proteasome (prosome, macropain) subunit, betatype, 9 (large multifunctional protease 2) (PSMB9), transcript variant 163 42680 AW449970.1 GE80314 DISCOVERY 1616.487305 42.0285644538.46163498 proteasome (prosome, macropain) 26S subunit, ATPase, 4(PSMC4), transcript variant 1

This experiment revealed the facts described below, and it was clarifiedthat the artificially synthesized peptide also significantly influencedthe expression of human cancer-related gene to induce cancer, as in thepeptide derived from the original cell line.

A. Enhanced Gene

1. Oncogene

Expression of oncogenes of cranial neuroblastoma, lung cancer, parotidgland tumor, breast cancer, colon cancer, renal cancer, ovarian cancer,melanoma, T-cell leukemia, B-cell leukemia, and so on were enhanced inthe range of 47 to 1.7 times the baseline expression thereof. Expressionof carcinogenic genes such as RAS, RAB, v-myc, and mab were alsoenhanced in the range of 6.7 to 1.7 times the baseline expressionthereof. Additionally, expression of cell growth factor such as aplatelet-derived growth factor was enhanced.

2. Immune System Gene

Expression of TNF, which is an inflammatory factor, was not enhanced,but expression of most of important factors as an operating factor forthe humoral immunity mechanism, which is the opposite side of anti-tumorimmunity, was enhanced.

B. Suppressed Gene

1. Cancer-Suppressing Gene

Every cancer-suppressing gene was significantly suppressed: Expressionof heat shock protein relating to cancer antigen-induction wassuppressed in the range of 1/461 to 1/116 of the baseline expressionthereof, and expression of a tumor rejection antigen was suppressed inthe range of 1/86 to 1/81 of the baseline expression thereof. Expressionof a gene relating to inhibition of metastasis of breast cancer wassuppressed to 1/60, expression of a gene relating to inhibition ofleukemia was suppressed to 1/43, and expression of RAS suppressorprotein gene was suppressed to 1/43.

2. Immune System Gene

The expression of genes relating to anti-tumor immunity was highlysuppressed as follows:

suppression of a gene relating to construction of thymus tissues orproduction of a thymic hormone;

suppression of a function and a construction factor of T-cell which isimportant in inhibition of cancer;

suppression of a function and construction of NK-cell;

suppression of production of a factor for killer-cell-secretion;

suppression of an apoptosis factor relating to cellular suicide;

suppression of a factor relating to cell adhesion in cell apoptosis;

suppression of production of antineoplastic interferon; and

suppression of expression of a cell organelle, an enzyme system, and amajor histocompatibility complex (MHC) relating to induction of a cancerantigen.

(b) The peptide obtained in Example 3 was added to a culture medium (10to 100 μg peptide/1×10⁷ monocytes) for human normal peripheral bloodmonocytes extracted from subjects shown in Table 5 below, and they werecocultured for 7 hr. Then, total mRNA was extracted from the monocytes,and the amount of the total mRNA was measured and compared to that ofthe total mRNA before the treatment with the peptide. Table 5 shows theresults.

TABLE 5 Effect of the artificially synthesized peptide on total mRNAamount of peripheral blood monocytes pre post total RNA total RNA post/reduction NAME (μg/ml) (μg/ml) pre (%) ratio (%) H.S. 45.875 0.958 2 98Healthy subject (smoker) M.S. 20.779 9.188 44.2 55.8 Healthy subject(smoker) E.E. 69.622 7.735 11.1 88.9 Healthy subject (side-stream smoke)S.G. 61.145 16.562 27 73 Healthy subject H.T. 66.804 14.742 22 78Healthy subject (side-stream smoke) M.O. 58.076 19.551 33.6 66.4 Healthysubject (side-stream smoke) M.A. 52.318 17.681 33.7 66.3 Healthy subjectK.K. 8.078 1.757 21.7 78.3 Cancer patient received chemotherapy andradiotherapy Samples are equivalent to 1 × 10⁷ peripheral bloodmonocytes. Treatment time with the peptide: 7 hr The processes forcollecting total RNA are the same as those in Example 4.

With referred to the results shown in Table 5, the total mRNA in H.S.and E.E. were significantly decreased. H.S. was an addicted smoker whohas smoked for more than ten years. With respect to M.S., the reductionratio of total mRNA was low, but the pre-total mRNA amount itself wasalso low, which has been caused by smoking over a long period of time.E.E. was not a smoker, but father of E.E. was an addicted smoker.Therefore, it was assumed that the decrease in the total mRNA was causedby passive smoking over a long period of time. Both H.T. and M.O. werepassive smokers, and the reactivity to the peptide was accelerated inproportion to degree of the passive smoking (H.T.>M.O.). In K.K., thereduction ratio was low and the pre-total mRNA amount was significantlysmall. This subject was a cancer patient who has already received ananticancer agent and irradiation at the maximum possible dose.Therefore, the general gene translation was significantly impaired;which caused the result entirely different from that in healthysubjects. The results shown in Table 5 show that the cancer diagnosiscan be efficiently performed by analyzing the changing ratio of totalmRNA amounts before and after the treatment with the peptide of thepresent invention.

Example 5 Detection of the Peptide of the Present Invention in CancerCells and Influence of the Peptide on Normal Cells

In order to examine influences at a gene level of the peptide on normalcells, RT-PCR was performed by adding the peptide artificiallysynthesized in Example 3 to the normal cells.

(Method and Result)

(1) Normal cells were treated with the artificially synthesized peptidefor 7 hr as follows:

No. of Cells Peptide Amount Kidney mesangium cell: 1.25 × 10⁶ 12.5 μg(ACBRI-1376; Applied Cell Biology Research Institute) Skin fibroblastcell:  4.0 × 10⁶ 40.0 μg (Catlog2F0-C25; Cell Systems) Pancreaticepithelial cells: 1.05 × 10⁷ 105.0 μg  (ACBRI515; Applied Cell BiologyResearch Institute) Peripheral blood monocytes: H.S. 1.25 × 10⁷ 12.5 μgM.S. 1.20 × 10⁷ 12.0 μg E.E. 1.40 × 10⁷ 14.0 μg (Kidney mesangium cells,skin fibroblast cells, and pancreatic epithelial cells: 100 μgpeptide/1.0 × 10⁷ cells, peripheral blood monocytes: 10 μg peptide/1.0 ×10⁷ cells)

Then, total RNA was extracted from the above-treated normal cells,untreated normal cells, and squamous-cell carcinoma cells by using anRNA extraction kit (QIAGEN). The numbers of the treated cells and theuntreated cells were the same.

No. of Cells RNA Amount Kidney mesangium cell: 1.25 × 10⁶ 30 μg(ACBRI-1376; Applied Cell Biology Research Institute) Skin fibroblastcell:  4.0 × 10⁶ 30 μg (Catlog2F0-C25; Cell Systems) Pancreaticepithelial cells: 1.05 × 10⁷ 30 μg (ACBRI515; Applied Cell BiologyResearch Institute) Peripheral blood monocytes: H.S. 1.25 × 10⁷ 60 μgM.S. 1.20 × 10⁷ 60 μg E.E. 1.40 × 10⁷ 60 μg Cervical carcinoma cells(HeLa): 1.60 × 10⁷ 60 μg

Reverse transcription was performed by using 30 μg of each extractedtotal RNA to generate cDNA pools.

PCR was conducted using each of the cDNA pools as templates.

The condition for the PCR was as follows:

PCR buffer: 6 μl

dNTP: 2 μl

Primer Fw (11 amino acids or 7 amino acids): 1 μl

Primer Rv (oligo dT): 1 μl

dH2O: 9.5 μl

taq: 0.5 μl

Sample (RT products): 10 μl

(Total: 30 μl)

PCR: 45 cycles

Initial denaturation: at 94° C. for 5 min

Denaturation: at 94° C. for 30 sec

Annealing: at 55° C. for 30 sec

Extension: at 72° C. for 1 min

(2) Each of the PCR products was applied to electrophoresis and detectedby using a fluorescence label.

(a) FIGS. 2A and 2B show the results when the full-length peptide (11amino acids) of the present invention was used as the primer in the PCR.

The results are as follows:

(i) Lanes 2 and 3 in FIG. 2A show the results of original UTC-8 cellsamples which were not treated with the peptide of the presentinvention. The original UTC-8 cell samples stably expressed the gene ofthe preset invention.

Lane 4 in FIG. 2A shows the result of a cervical carcinoma cell sample,wherein a smear was observed. This means that the sample includesvarious sizes of genes which are complementary to the gene encoding thepeptide of the present invention. Namely, the expression pattern of thegene was significantly changed in the cervical carcinoma cell sample bythe treatment with the peptide of the present invention. Therefore,samples showing such a reaction pattern are thought to have a tendencyof canceration.

(ii) On the basis of the results above, samples on each lane wereexamined. In some samples (Lanes 5 and 6 of FIG. 2A, lanes 4 and 5,lanes 6 and 7, and lanes 8 and 9 in FIG. 2B), the original stable genepattern was changed to a pattern having a broadened smear by thetreatment with the peptide of the present invention. Such samples weredetermined to have a tendency of canceration. Actually, the blood sampleof the lanes 5 and 6 of FIG. 2A was obtained from a young man who hadsmoked for more than 10 years. Though the samples were normal cells,they had a high sensitivity to the peptide. It is thought that such ahigh sensitivity is caused by that these cells were, as in the peptideof the present invention, extracted from epithelial cells and werederived from a tissue in an early stage. Therefore, the gene expressionis not fixedly stable, and the unstable gene expression is involved inthe acuity sensitivity against a highly disturbing factor from theoutside such as the peptide of the present invention.(iii) On the contrary, in the sample of lanes 2 and 3 of FIG. 2B, genemutation was hardly induced even if the sample was treated with thepeptide of the present invention. This sample is thought to have acharacteristic to rarely get cancer. Actually, the sample was obtainedfrom a healthy young man in twenties.(b) FIGS. 3A and 3B show the results when a DNA encoding an amino acidsequence (7 amino acids) at positions 1-7 of the peptide of the presentinvention was used as a primer in the PCR.

The results are as follows:

(i) Smears were observed in an UTC-8 cell sample shown in lane 3 of FIG.3A and in a cervical carcinoma cell sample shown in lane 4 of FIG. 3A,as in the results when the full sequence was examined in theabove-mentioned (a).

(ii) In the samples of lanes 2 and 3, lanes 4 and 5 of FIG. 3B, smearswere not observed even if the samples were treated with the peptide ofthe present invention, and normal patterns were observed.

(iii) In the samples of lanes 5 and 6, lanes 7 and 8 of FIG. 3A andlanes 6 and 7, lanes 8 and 9 of FIG. 3B, gene expression patterns weresignificantly changed before and after the treatment with the peptide ofthe present invention.

As shown above, it was clarified that a tendency of canceration innormal cells can be determined by comparing changes in gene patternsinduced by treating the cells with the peptide of the present invention.

In particular, when the PCR was performed by using the 11-amino acidprimer (full sequence), a difference in the band patterns was observedin the peripheral blood sample (H.S.) and the skin fibroblast sample,and a difference in a position having a high concentration was observedin the kidney mesangium sample and the pancreatic epithelial sample,though it was a smear. When the PCR was performed by using the 7-aminoacid primer (partial sequence), the band pattern of the pancreaticepithelial sample treated with the peptide for 7 hr was similar to thatof the UTC-8 cell sample used as a control, and a difference in the bandpatterns was observed in the pancreatic epithelial sample by thetreatment with the peptide for 7 hr.

A possible explanation for causes of such significant effects is thatthe UTC-8 cell is a cancer cell of epithelium and the peptide of thepresent invention is derived from UTC-8 cell line.

Therefore, epithelial cells are the most suitable samples for themeasurement of a tendency of canceration using the peptide of thepresent invention, but it was proved that non-epithelial cell samplesincluding peripheral blood, which can be most easily obtained inclinical practice, were useful for the diagnosis of a gene change in thefull sequence.

Example 6 Exhaustive Gene Analysis of 55000 Genes by Using a DNA ChipSystem (Codelink System)

Characteristics of the peptide of the present invention were clarifiedby conducting the analysis described below in order to examine effectsof the peptide on gene expression of normal cells.

(1) Preparation of Target cRNA

Normal lymphocytes were treated with the peptide of the presentinvention as in Examples 4 and 5, and the gene expression was comparedto that of untreated lymphocytes by using a DNA chip system (CodelinkSystem). In this examination, cRNA derived from bacteria was used as apositive control. Total RNA was extracted by using the QIAGEN RNAextraction kit used in Example 5. The total RNA (5 to 10 μg) wassubjected to reverse transcription using an oligo-dT primer having a T7RNA polymerase promoter region as a reverse primer to generate a pool ofcDNAs complementary to the mRNAs. The condition for the reversetranscription was the same as that in Example 5. After second-strandcDNA synthesis, the treatment with T7 RNA polymerase and biotinylatedrNTPs was performed in vitro transcription (IVT) reaction at 37° C. for14 hr to obtain biotin-labeled cRNA. The amount of the cRNA was measuredwith a spectrophotometer to confirm at least 10 μg of the cRNA.

(2) Hybridization to Codelink Slide

The cRNA (10 μg) obtained in the above (1) was fragmentized and mixedwith hybridization buffer, and then thermally denatured. This mixturesolution was poured into a Codelink slide on which 55000 genes wereimmobilized, and a hybridization reaction was performed at 37° C. for 18hr using an INNOVA 4080 shaker.

(3) Staining and Detection

The hybridization chamber was removed and the plate was washed at 46° C.for 1 hr. The hybridization was detected with Cy5-streptavidin (1:500dilution, at room temperature for 30 min). The plate was further washedat a room temperature for 5 min 5 to 6 times and then washed with 0.05%Tween 20. The plate was dried by using a centrifuge for titer plates.

(4) The plate was scanned with an Arra WORK scanner with a PMT value of600 at a resolution of 10 μm.

(5) The result was analyzed by exclusive software for the Codelinkanalysis. The software used was 1. Batch Submission 2 and 2. CodelinkExpression 2.

Cancer-inducing expression status of cancer-related genes was observedin this experiment too, as in the results shown in Tables 1, 2, and 3.

Example 7 Examination of Antibody Against the Peptide for CytotoxicActivity on Human Cancer Cells

Monoclonal antibody (prepared by using BALB/c mouse) against the peptideof the present invention was examined for cytotoxic activity on varioushuman cancer cell lines shown in FIG. 4: Each cancer cell line wasseeded in a 96-well plate at 1×10⁴ cells/well, and cultured in 5% CO₂for 4 days to confirm that the cancer cells proliferated and adhered tothe bottom surface of the plate so as to cover more than 80% of thesurface area. The culture medium was removed by aspiration, and then 30μl of the monoclonal antibody was added to each well under ice-coolingso that the surfaces of the cancer cells were brought into contact withthe culture medium containing the monoclonal antibody. Then, the cancercells were cultured under ice-cooling for 1 hr. As a control, eachcancer cell line was also cultured in PBS buffer not containing themonoclonal antibody. Cytotoxic activity of the monoclonal antibody wasexamined by comparing the cancer cells treated with the monoclonalantibody with those of the control, and viability of the cell wasdetermined by Trypan Blue staining. In the control, no substantialdetachment of the cells from the bottom surface of the plate wasobserved; hence, cytotoxicity was not observed. On the contrary, in thecancer cells treated with the monoclonal antibody against the peptide ofthe present invention, detachment of the cells was observed in cancercells shown in FIG. 4; hence, cytotoxic activity of the monoclonalantibody was observed. In addition, the detachment of the cancer cellswas immediately induced by the addition of the monoclonal antibody, andthe cancer cells were killed by the activity of the monoclonal antibodywithin only one hour. The photograph in the right of FIG. 4 shows humanpancreatic cancer cells stained with FITC-labeled monoclonal antibodyNo. 1 of the present invention. These human pancreatic cancer cells diedafter this treatment.

Example 8 Immunosuppressive Agent Reducing Rejection in Transplantation

The experiment using the DNA chip proved that the peptide (SEQ ID NO: 1)had an ability to activate a cancer-related gene, and further suggestedclinical usefulness of the cellular immunity-inhibition activity of thepeptide. Then, the cellular immunity-inhibition activity of the peptidewas examined from the viewpoint of activity for preventing rejection inskin transplantation.

The peptide of the present invention was administered to mice who havereceived skin transplantation according to the schedule shown in Table6.

TABLE 6 Days from Administration Transplantation Transplantation ofPeptide −3 Administration −2 Administration −1 Administration 0Operation 1 2 Administration 3 Administration 4 5 6 7 Administration 8 910 11 12 13 14 Administration 15 16 Subject: skin of the back of aC57Black/6N mouse was transplanted to the back of a BALB/c mouseAdministration route: subcutaneously administered into the neckAdministration amount: 50 μg peptide/0.1 ml/mouse Number of the subject:6

The results show that, in the cross-transplantation of skin of the backof a C57Black/6N mouse to the back of a BALB/c mouse, the rejection inthe skin transplantation was suppressed and skin graft survival wasprolonged by intermittently administering the peptide at a very lowamount of 50 μg peptide/0.1 ml/mouse through a subcutaneous route whichis a most gentle administration (see the progress shown by the graph inFIG. 5). Namely, in the control, skin rejection occurred 7 days afterthe skin transplantation and the transplanted skin was rejected and waslost within 10 days after the transplantation in all 6 mice as shown ina photograph (control G1) of mice at bottom right in FIG. 5. On thecontrary, in the mice administered with the peptide, skin rejectionoccurred 12 days after the transplantation; which was a 5 day delay.Furthermore, the transplanted skin of one mouse was survived for 14 daysafter the transplantation as shown in a photograph (treatment G1) atupper right in FIG. 5. Though the transplanted skin was lost in all miceon the 15th day from the date of the transplantation, a delay in therejection in skin transplantation was observed.

Therefore, it was proved that the peptide can be used as animmunosuppressive agent by investigating administration dosage and routeto increase efficiency thereof.

1. A diagnostic kit for determining tendency of canceration ormalignancy of cancer, wherein the kit comprises (i) a peptide having anability to activate a cancer-related gene, wherein the peptide isobtained from cell membrane surfaces of human squamous-cell carcinomacells and comprises the amino acid sequence represented by SEQ ID NO: 1,and (ii) a polynucleotide comprising a nucleotide sequence encoding theeleven contiguous amino acids of the amino acid sequence of a peptidehaving an ability to activate a cancer-related gene, wherein the peptideis obtained from cell membrane surfaces of human squamous-cell carcinomacells and consists of the amino acid sequence represented by SEQ IDNO:
 1. 2. The diagnostic kit according to claim 1 further comprising atleast one marker gene selected from the group consisting of: Rasoncogene family; v-crk avian sarcoma virus CT10 oncogene homolog lactatedehydrogenase B; Placental growth factor; Interleukin 8; MAS1, activatorof S phase kinase; v-raf; v-fms; v-rel; IT-STC; GRO1; Hepatoma-derivedgrowth factor; Vascular endothelial growth factor; Bone morphogenicprotein 3; Squamous-cell carcinoma antigen recognized by T cell;Interleukin-1 beta; Conserved gene amplified in osteosarcoma; andLymphoid blast crisis oncogene.
 3. A diagnostic kit for determiningtendency of canceration or malignancy of cancer, wherein the kitcomprises (i) a peptide having an ability to activate a cancer-relatedgene, wherein the peptide is obtained from cell line UTC-8 (FERMBP-08611) established from human squamous-cell carcinoma; and thepeptide shows a detectable peak at a detection wavelength of 214 nm insamples collected during gel filtration of an extract of the cell line,and (ii) a polynucleotide comprising a nucleotide sequence encoding theeleven contiguous amino acids of the amino acid sequence of a peptidehaving an ability to activate a cancer-related gene, wherein the peptideis obtained from cell membrane surfaces of human squamous-cell carcinomacells and consists of the amino acid sequence represented by SEQ IDNO:
 1. 4. The diagnostic kit according to claim 3 further comprising atleast one marker gene selected from the group consisting of: Rasoncogene family; v-crk avian sarcoma virus CT10 oncogene homolog lactatedehydrogenase B; Placental growth factor; Interleukin 8; MAS1, activatorof S phase kinase; v-raf; v-fms; v-rel; v-src; GRO1; Hepatoma-derivedgrowth factor; Vascular endothelial growth factor; Bone morphogenicprotein 3; Squamous-cell carcinoma antigen recognized by T cell;Interleukin-1 beta; Conserved gene amplified in osteosarcoma; andLymphoid blast crisis oncogene.
 5. The diagnostic kit according to claim1, wherein the peptide (i) in the kit consists of the amino acidsequence represented by SEQ ID NO: 1.